Na+, the predominant cation in the extracellular fluid, is involved in numerous physiological and pathophysiological processes. Arterial myocytes, like most other types of cells, normally maintain a low intracellular Na+ concentration. The Na+ gradient is involved in the regulation of intracellular Ca2+ concentration [Ca2+]i and pH, contractility, cell volume and membrane potential, in the distribution of numerous solutes, and many other functions. For vascular smooth muscle cells there is little quantitative information available regarding the intracellular Na+ concentration ([Na+]i), and the temporal and spatial changes in [Na+]i, such as those that are evoked by vasoconstrictors and vasodilators. We have obtained evidence that various agonists evoke a common Na+ response, namely a sustained elevation of [Na+]i. The goals of this project are to investigate: whether such an increase in [Na+]i is a general phenomenon for arterial myocytes; what the mechanisms underlying these effects are, and what implications arise in [Na+]i, evoked by different stimuli, has on [Ca2+]i, the main determinant of muscle contraction. In this project I will: 1) Determine changes in [Na+]i evoked by selected vasoconstrictors, growth factors and vasodilators. Digital imaging analysis of cells loaded with the Na+-sensitive fluorescent dye, SBFI, will be used to determine temporal and spatial changes in [Na+]i. 2) Determine whether agonist-induced changes in [Na+]i are due to net agonist-induced changes in Na+ influx and/or Na+ efflux. 3) Elucidate the mechanisms of acute agonist-induced regulation of the Na+ pump, the main determinant of [Na+]i. The main emphasis of this part of the project is placed on testing the hypothesis that the Na+ pump activity is regulated, in part, by phosphorylation/dephosphorylation processes. Effects of intracellular kinases (e.g., protein kinase C) and phosphatases (e.g., phosphatase 1) on the Na+ pump activity and phosphorylation status will be determined and compared with the effects of the physiological agonists. The Na+ pump activity will be evaluated in single cultured cells (measurements of SBFI fluorescence) and in native tissue (i.e. arterial rings; measurements of 86Rbuptake). To determine the phosphorylation status of the Na+ pump, the enzyme will be immunoprecipitated, using specific antibodies, from 32P04 labelled cells. 4) Compare the regulation of [Na+]i in activated myocytes from conduit and muscular arteries. 5) Demonstrate the critical role of the rise in {Na+]i in augmentation of the amount of stored Ca2+. The accumulation of Ca2+ in stores and its release will be evaluated using digital imaging analysis of cells loaded with Ca2+-sensitive fluorescent dyes, chlortetracycline and fura-2, respectively. The results of these studies should provide new information about the regulation of Na+ in arterial myocytes and its relation to the regulation of Ca2+. Moreover, these findings may be useful in elucidating pathophysiological processes, such as those associated with (Na+-sensitive) hypertension or ischemia.